Project Summary The long term goal of the application is to understand why and how lipoproteins from Firmicutes undergo modifications. Lipoproteins are membrane associated globular proteins anchored to the bacterial membrane surface through a lipidated N-terminal cysteine residue. They are ubiquitous cell envelope structures found in both gram positive and negative bacteria, accounting for 1-5% of all genes in a typical bacterial genome. Lipoproteins play roles in nearly every aspect of bacterial cell envelope physiology, from nutrient acquisition to mediating cellular contacts. Lipoproteins are also important during the detection and mounting of initial immune responses to clear bacteria infections. Due to their multiple essential cellular roles, abundance, universal distribution, and their unique and highly conserved structure, innate immunity detects the presence of bacteria through binding lipoproteins using Toll-like receptor 2 (TLR-2) complexes. TLR2 binds lipoproteins through forming heterodimers, using either TLR-1 or TLR-6 depending on the state of N-acylation. As such, understanding how and why certain bacteria utilize lipoprotein structural variations is pertinent to both fundamental bacterial physiology and infection biology. In particular, this application studies the role of N-acylation in Enterococcus faecalis. There is emerging evidence that lipoproteins are differentially N-acylated amongst bacteria in the Firmicutes phylum. A class of integral membrane N-acylating proteins named Lit was identified in E. faecalis that makes lyso- form lipoproteins in this organism. The lyso- form of lipoprotein has a unique acyl chain distribution pattern. Whereas the diacylglyceryl form has both acyl chains on the glyceryl residue, and the triacyl form has these two acyl chains plus a third N-terminal acyl chain connected through an amide bond to the ?-amino cysteine group, the lyso-form has a single acyl chain on both the N-terminus and glyceryl unit. The lyso- structure suggests an acyl chain is removed from the diacylglyceryl unit and transferred to the N-terminus to form the lyso- structure or that there are lipoprotein esterases working concert with Lit. The application aims to reconstitute Lit activity by recombinant expression so as to test the proposed intramolecular transferase using specifically labeled diacylglyceryl lipoprotein substrates. The products will be characterized by MALDI mass spectrometry to determine acyl chain donor origin. A second aim is to test environmental stress condition in order to gain insight into the biological role for N-acylation using the E. faecalis/?lit isogenic paired strain set. The third aim is to probe how Lit affects TLR-2 signaling, as preliminary data has suggested lyso-form lipoproteins bind in more complex ways to TLR2 than their triacyl- and diacyl-glycerol congeners. The final aim is to develop functional assays to uncover other lipoprotein N-terminal modifying enzymes.